Ectopic expression of Sfla Or67b3 in Or22a OSNs or Or67b OSNs conferred behavioral responses to BITC in D. melanogaster. The behavioral responses of D. melanogaster flies expressing different Or67b transgenes were tested in dual-choice assays (odorant vs. mineral oil) and experiments were conducted and analyzed as in figure 5 but flies were starved 24 h previous to testing. (A) Responses of flies expressing Dmel Or67b or Sfla Or67b3 in Or22a OSNs lacking its cognate Or and tested with BITC 1:1,000 vol/vol versus mineral oil. The two parental control lines (first two groups) and flies expressing Dmel Or67b (third group) were not attracted or repelled by BITC (binomial tests, P > 0.05 in all cases), whereas flies expressing Sfla Or67b3 were attracted to the odorant (*P < 0.05). Importantly, parental genetic control lines retain olfactory attraction toward apple cider vinegar, a potent D. melanogaster olfactory attractant and activator (supplementary fig. 10, Supplementary Material online). These results show that ITCs can evoke olfactory behavioral responses when Sfla Or67b3 is expressed in the D. melanogaster Or22a olfactory pathway, an important circuit for detection of host odorants in many drosophilid species (Dekker et al. 2006; Linz et al. 2013; Mansourian et al. 2018). (B) Same as (A), but flies expressed Dmel Or67b or Sfla Or67b3 in Or67b OSNs. Note that flies have the endogenous Or67b expressed in OSNs, in addition to the transgene. As in (A), only flies carrying the Sfla Or 67b3 transgene were attracted to BITC (*P < 0.05, third group). (C) Responses of D. melanogaster expressing a silencer of synaptic activity (Kir2.1) in Or67b OSNs (third group), along with the responses of the two parental genetic control lines (first and second group) in tests with acetophenone 1:50,000 vol/vol, a strong Dmel Or67b activator (Münch and Galizia 2016; fig. 3). Genetic control flies showed a trend for attraction (0.05 < P < 0.08) to low concentrations of acetophenone, whereas flies with Or67b OSNs silenced lost attraction and were instead repelled by the odorant. These behaviors mirrored those of wild-type D. melanogaster flies tested with low and high concentrations of acetophenone, respectively (supplementary fig. S11, Supplementary Material online; Strutz et al. 2014). In (A–C), transgenes are indicated to the left; gray-shaded bars serve to visualize that the proportion of tests with flies orienting toward that arm of the y-maze is significantly different (P < 0.05) from random.